Bacterial expression of a designed single‐chain IL‐10 prevents severe lung inflammation

Abstract Interleukin‐10 (IL‐10) is an anti‐inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL‐10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non‐pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL‐10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single‐chain IL‐10 (SC‐IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL‐10 WT, the designed SC‐IL10 molecules increase the effective expression in MPN four‐fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC‐IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti‐inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy.

The MutSC1 and MutSC2 (currently in Figures 3B-D) should not be shown before the design of MutSC1 and MutSC2 (currently Figure 4). This anachronistic presentation makes the manuscript more difficult to follow.
The description of how MutSC1 was designed is insufficient to understand both the molecule itself and the methods that were used to design it. For example, there is discussion of a linker sequence but where that sequence is introduced in the molecule is not described. As a second example, I am not familiar with "side chain repair" or why that would be utilized to design MutSC1. Figure 4 provides some of the information, but does not completely address these issues. not secrete as much IL-10 ORF as the non-attenuated strain.
Reviewer #2: In this work, Montero-Blay et al. propose an engineered living therapeutic aimed at addressing inflammatory conditions in the lung. For this, they create an attenuated strain of M. pneumoniae that expresses engineered versions IL-10. While single-chain IL-10 versions had been engineered previously, the authors rationally designed new versions of single-chain IL-10 with enhanced properties that might prove useful to address the challenges inherent to the lung environment and the use of MPN as a therapeutic chassis. The authors then use a mouse model of P. aeruginosa infection to evaluate the efficacy of the engineered bacteria in ameliorating inflammation in the lungs and present data that suggests their new versions of IL-10 display enhanced anti-inflammatory effect.
-I have a major concern about the data derived from the mouse studies. The authors do not report the exact number of animals used per group nor whether they performed (more than one) independent experiments to support the validity of the data. They only present a vague reference in the methods section that states: "Infections were performed in groups of at least 5 mice per strain (n {greater than or equal to} 5)". Without the sample size information, it is difficult to assess the significance of the data and it undermines the interpretation of the results. The authors should state the exact number of mice used in each group and present the data as individual data points along with the appropriate measure of dispersion. Mouse studies that support major claims of the work should be performed at least twice in independent experiments.
-Structuring and writing of the manuscript should be improved. The last paragraph of the introduction section presents results and interpretations before the data is shown. Methods and Discussion sections also report experimental results. Why is figure 4 presented in the text before addressing Fig 3C and D? Please reorganize text and figures to follow the logical flow of a scientific article.
-"Afterwards, we mutated the Phe after N157 to Asn to improve binding to R1 (this results in NGGLD as the inserted sequence; Fig. 4A); this improved the overall stability of the SC molecule as well as its binding to R1." Please state the metric used and present the data that supports the claim of improved stability.
-"Regarding the CV8 strain, we could see a minor inflammatory response when it was used alone (Fig. 6C), ...". Please describe what constitutes a "minor" inflammatory response. This sort of qualification seems subjective.
-"...as a drug factory that is easy to remove with antibiotics once its action is completed.". This statement should be revised. No data is presented supporting the efficacy of antibiotics in removing CV8 and related strains from the lungs. Antibiotic treatment should not be considered as biocontainment strategy due to the undesired effects that it might have i.e., emergence of resistant variants, dysbiosis, etc. Further discussion of an effective biocontainment strategy for this lung-targeted living therapeutic should be included.

Reviewer #3:
The manuscript submitted by Ariadna Montero-Blay and colleagues is divided into two main themes: 1) the rational engineering of IL-10 using computational protein design tools, and 2) the development of the Mycoplasma pneumoniae CV8 live biotherapeutic cell chassis and its activity in a mouse model of lung infection. Different versions of IL-10 were prepared including surface mutations and single-chain conformations (given that IL-10 forms intra-monomer disulfide bonds). The level of secretion by the CV8 chassis and the activity of some of the IL-10 variants were increased in vitro and displayed nice anti-inflammatory properties in mice infected with Pseudomonas.
The manuscript is a nice demonstration of the potential of live biotherapeutics and how rational engineering of active molecules can potentiate their effects. The work is following a recently published article in Molecular Systems biology in which a M. pneumoniae live biotherapeutic chassis was presented. The key difference is the molecule (IL-10) that was secreted and the organ that is treated (lung) while the previous work was focussing on biofilm and catheters.
Overall, I found the work to be interesting and well performed. However, some sections can be confusing and would require rewording or clarifications. I would encourage the authors to use line numbers in future manuscripts to facilitate the review process.
1) Rewording a few sentences would enhance clarity and avoid misinterpretation.
-Activity of IL-10 derivatives; a few sentences suggest that the activity of the single-chain IL-10 shows a 60-fold increase in activity (abstract line 9; page 13, line 12. This can be misleading as the result is a combination of increased secretion by Mycoplasma pneumoniae and better activation of the receptor. There is no head-to-head comparison of purified single-chain IL-10 derivative with WT IL-10 at the same concentration that would support this conclusion. These sentence should be reworded to avoid any misunderstanding.
-page 3, line 21. The sentence about the selection of a live biotherapeutic chassis with regards to the organ to treat could be reworded to improve syntax and clarity.
2) the information in certain Supplementary Tables is difficult to understand without additional details: -page 12, second paragraph, lines 7-8: it is not obvious to me that the lung is more challenging than intestine for live biotherapeutics. The authors could clarify their thoughts. I believe they meant that fewer bacteria are present in this organ but there are other factors to consider and is not as simple as this.
-Supplementary Table 1: It is difficult to understand why a correction factor was applied and how this number was determined. This should be explained.
-Supplementary Table 2: why is the format of the data different in the "sulfide MutSC1" tab compared to "Sulfide WT IL-10" and "Sulfide MutSC2"? Minor: -page 11, last line: 2 ug instead of 2 ugr.
-page 15, first sentence of the last paragraph: ATCC instead of ATTC.
-page 25, line 6. Bmax should be defined at first use.
-Supplementary Table 6: the "Inflammatory makers" tab should be "Inflammatory markers" -Supplementary Table 10: why is only the amino acid sequence provided for IL-10 derivatives? DNA sequences would ideally also be reported.

Reviewer #1:
We would like to thank you for your critical revision of our manuscript ("Bacterial expression of a designed single-chain IL-10 prevents lung severe inflammation"). We have taken your comments in consideration and have now addressed all the issues you raised. (Note that new changes in the manuscript text shown here are highlighted in blue).
Bacteria engineered to secrete anti-inflammatory cytokines have applications as nextgeneration therapeutics. The authors have previously engineered the pathogen M.
pneumoniae as a protein secretion chassis for therapeutic applications in the lungs.
Here, the authors substantially advance their previous work by i) engineering M. pneumoniae to secrete the potent homodimeric anti-inflammatory cytokine IL-10, ii) using a structure-guided approach to identify mutations that increase IL-10 activity, iii) engineering novel single-chain IL-10 mutants that are secreted by M. pneumoniae to higher levels and have improved activity, iv) developing a novel P. aeruginosa mouse model of lung inflammation in order to test the therapeutic efficacy of their strains, vi) engineering a novel M. pneumoniae strain with substantially reduced pathogenicity to use for therapeutic purposes. They provide evidence that their approach results in strong attenuation of inflammation in the lungs. These results could be advanced toward new therapeutics for inflammatory diseases of the lungs, including pneumoniae or viral (e.g. SARS-CoV2) infections. Their single-chain IL10 molecules could also have other therapeutic applications.
The manuscript is innovative and impactful. I believe that it is appropriate for publication in Molecular Systems Biology, pending a number of improvements to the presentation and several key additional experiments.

Specific comments
Please add line numbers.

Added
The IL-10 functionality experiment in Figure 1  We thank the reviewer for this comment. The main point of this experiment was to demonstrate that IL-10 expressed by Mycoplasma is active and capable of modulating the response by macrophages. In the submitted data, results of four independent donors were presented as bars in a plot. These data were not normalized; rather, mean fluorescence intensity (MFI) values for the donors (n=4) are shown. As the data were not normalized, the statistical difference of controls and treated cells with commercial and Mycoplasma-expressed IL-10 was not significant for many of the biomarkers, even though all donors showed similar trends of up-and downregulation of them.
To solve this issue, and to address the reviewer's comment, we have made a new  Table EV1).
The results now reveal a significant effect of the Mycoplasma secreted IL-10 (WT_IL10 supernatant) on the anti-inflammatory CD163, CD16, and PDL1 markers, as well as a tendency for MerTK. In addition, we also observed the statistical significance of the downregulation of the pro-inflammatory markers MHC-II and CD86.
No information is given in the main text, figure legend, nor methods section about how the bacterial supernatant-human cell treatment experiment was done. A detailed methodological description of this experiment should be added.
We have now restructured the Methods and provided further information. Specifically, the experimental details given in the 'Blood monocyte assays' section are now organized into two following subsections: "Blood monocyte Isolation" and "IL-10 stimulation and analysis by flow cytometry". This modification appears in the manuscript in lines 420-454.
From the main text, it sounds like only a single mutation was introduced into IL-10 Mut1 and Mut2. The descriptions of the design of these mutants should be clarified.
We apologize for the confusion. Mut1 and Mut2, as well as the rest of the mutants, are fully described in Table EV9. We have now added the amino acid compositions, DNA sequences, and specific details for each mutant.
The specific set of mutations introduced in IL-10 for Mut1 and Mut2 are the following: The difference between these two mutants is at position 31, where Mut1 has an Arg and Mut2 a Lys. This information is included in lines 121-123 as follows: Using the selected mutations, we modeled two multiple mutants (Mut1 and Mut2), which contain the mutations D28E,S31K/R,N45S,TL155M; note that these differ at position 31, whereby the WT Ser31 is mutated to Lys in Mut1 and Arg in Mut2 (Fig. 2).
I do not understand the statement "We checked that the mutations were independent of the structure used by...." on page 6. The authors are describing part of their high affinity IL-10 computational design procedure, but the approach is not clear. This statement should be clarified. This is a complex issue to describe. In order to be more precise, we have added the corresponding free energies for PDB=1j7v in Table EV3 and modified the paragraph in the revised version, which we hope is clearer.
"We verified that the differences in stability and binding energies were not the result of a particular conformation of the crystal structure that we used by computing the in silico variation of free energies of the multiple mutations of IL-10 in Mut1 and Mut2 using other experimental structures (IL-10 apo form: PDB 2ilk, 1.6 Å; IL-10 holo form with R1: PDB 1j7v, 2.9 Å; IL-10 holo form with R1&R2: PDB 6x93, 3.5 Å). We looked for changes in energy that affected IL-10 stability, interactions with R1 and R2 (when present in the PDB structure), and stability of IL-10 receptor complex (Table EV3)." (Lines 123-129) How many and which mutations were introduced into IL-10 to generate Mut3 is not clear (ambiguous) based on the main text description. This information should be clarified in the main text.
As stated in the previous point, we apologize for the confusion.
Mut 3 is the result of introducing R2-enhancing affinity mutations on top of those introduced in Mut2. Therefore, the composition of the mutant is D28E, S31K, N45S, T155M, N18I, N92I, K99N. This information is now clarified in the text in lines 130-133 and in Table EV9.
At the start of this work, no structure was available of IL-10 bound to R2. Therefore, we included mutations in Mut2 at some positions previously reported to potentially enhance the affinity of the interactions between IL-10 and R2 (e.g., N18I, N92I, and The scale of the axis in Figure 3a is wrong (EC-50 values of 10^10).
Thanks for pointing this out; we have now corrected this typo in the new version (new Fig 4A).
The claim that the measured affinity of MutM is lower than that of IL-10 ORF is not supported by the data ( Figure 3A). In particular, the two are statistically equivalent.
They mentioned that this mutant was less active than the IL-10 WT (stoichiometry of interaction of 1:2:2). We find a similar tendency (although not statistically significant,  Figures 3B-D) should not be shown before the design of MutSC1 and MutSC2 (currently Figure 4). This anachronistic presentation makes the manuscript more difficult to follow.
According to the reviewer's suggestion, we have switched the order of the Figures 3 and   4, and reassigned the references in the manuscript accordingly.
The description of how MutSC1 was designed is insufficient to understand both the molecule itself and the methods that were used to design it. For example, there is discussion of a linker sequence but where that sequence is introduced in the molecule is not described. As a second example, I am not familiar with "side chain repair" or why that would be utilized to design MutSC1. Figure 4 provides some of the information, but does not completely address these issues.
We have modified the single-chain IL-10 rational design section to make it more understandable and we have added the monomer descriptors in Figure 3 to accompany the changes (lines 387-395). Since we have grasped a peptide fragment from the ModelX database of peptide fragments to link the N-and C-termini of IL-10, we bring the original rotamers found in the structure from where the fragment was taken. These rotamers are not necessarily the same or they will require some small torsional moves in order to be accommodated in the context of the IL-10 structure. For this reason we use the RepairPDB FoldX command, to try to accomodate side chains of the fragment and of the surrounding residues of IL-10.
The authors report the efficacy of MutSC1 and MutSC2 by comparing their EC50 values to that of IL-10 ORF. However, the IL-10 ORF data is absent from the relevant main text figure (Figures 3C and D). The IL-10 ORF data should be plotted against the MutSC1 and MutSC2 data.
In Figure 3C and D, we plot the ratios in EC-50 between the designed IL-10 variants and IL-10 WT (IL-10 ORF) to indicate the fold improvement (The original data are presented in Table EV5). In order to clarify this information, we have modified the figure legend and axis and included the standard deviation in the figure to make this clear. We think this is the best way to normalize the results. However, if the reviewer thinks that we should put the original data we are happy to change the figure.
"One fold better" is not a common way to compare two data sets and it suggests that two things behave the same way. Perhaps the authors mean to say 100% greater?
We agree with the reviewer's suggestion and have now modified the text following this suggestion (line 340-341 and line 215).
In all cases, we found that MutSC2 had a 100% higher relative affinity than MutSC1.

As seen with HEK-Blue TM cells, MutSC2 was around 100% better than MutSC1
The authors do not characterize the secretion levels of IL-10 ORF, MutSC1 or MutSC2 from CV8. This experiment must be performed in order to demonstrate that their virulence-attenuated M. pneumoniae strain does not have compromised protein production or secretion capacity. This is particularly important, because the authors observe no anti-inflammatory effect from IL-10 using their attenuated chassis in their P.
aeruginosa model (Figure 6c). One possible explanation for this is that CV8 does not secrete as much IL-10 ORF as the non-attenuated strain.
We have added a new set of experiments that provide evidence for the similar protein secretion capacity of the CV8 and WT M. pneumoniae strains (new Figure 5, panel D).
We found no statistical significant differences when comparing expression of IL-10 WT and the MutSC1 and MutSC2 variants in the WT or CV8 strains.

We have added a new sentence in the manuscript in lines 249-250
No differences in protein capacity were observed between WT-expressing and CV8expressing strains ( Figure 5D).

Reviewer #2:
We would like to thank you for your critical revision of our manuscript ("Bacterial expression of a designed single-chain IL-10 prevents lung severe inflammation"). We We agree with this comment. We understand that this suggestion concerns the mice assay performed to test the immunomodulatory effect of IL-10 single chains MutSC1 and MutSC2. To address this comment: i) We repeated the assay with n=5 additional mice per group, and the new results confirmed our previous results. We have added this new experiment to the new Table   EV7 as well as to the new Figure 6 (whereby results from experiment 1 are indicated by circles, and experiment 2, by squares).
ii) Each mouse is represented as an individual point (see new Figure 6).

iii) We have now added the number of mice used in this study in the Material and
Method section, a Supplementary Table (new Table EV7) showing the mice used in the previous version (experiment 1) and the replica (experiment 2), and the criteria of exclusion. For the latter: 1) only animals with PAO1 and/or M. pneumoniae recovery (log >0.52) were considered; 2) animals with technical issues (i.e. contaminated plates) were excluded; and 3) for qPCR analysis, samples with total RNA concentration >100 ng/ul of total RNA extraction and ratio A260/A230 1.8-2.2 were considered.
iv) Considering that we are interested in the effect of the CV8 chassis coding for the designed IL-10 molecules, we showed the statistical comparison between the PAO1 + CV8 reference group and the PAO1 + CV8_IL10, PAO1 + CV8_MutSC1, PAO1 + CV8_MutSC2 and PAO1 + hIL10r groups in the new Figure 6. In addition, we included the sheet "p value" in the new Table EV7, in which we specified all the statistical values, including the comparison with the PAO1 + PBS, that present the same tendency.
We appreciate this comment because it has reinforced our previous statements and the quality of the work.
-Structuring and writing of the manuscript should be improved. The last paragraph of the introduction section presents results and interpretations before the data is shown.
Methods and Discussion sections also report experimental results. Why is figure 4 presented in the text before addressing Fig 3C and D? Please reorganize text and figures to follow the logical flow of a scientific article.
We agree with the reviewer's suggestion and have switched the order of the Figure 3 and Figure 4 and reassigned the references in the manuscript accordingly. The Figure   EV4 is now cited in the Results section. Also, we removed the sections that introduce results and interpretations from the introduction section.
-"Afterwards, we mutated the Phe after N157 to Asn to improve binding to R1 (this results in NGGLD as the inserted sequence; Fig. 4A); this improved the overall stability of the SC molecule as well as its binding to R1." Please state the metric used and present the data that supports the claim of improved stability.
We have modified the paragraph to explain that claim (lines 180-183) as follows: "Afterwards, we mutated the Phe after N157 to Asn to improve binding to R1 (this results in NGGLD as the inserted sequence; Fig. 3A); this improved the FoldXpredicted overall stability of the SC molecule, creating an intramolecular HBond with G160, rigidifying the inserted loop, and consequently its binding to R1 by stabilising the bound state." -"Regarding the CV8 strain, we could see a minor inflammatory response when it was used alone (Fig. 6C), ...". Please describe what constitutes a "minor" inflammatory response. This sort of qualification seems subjective.
We agree with this comment and have replaced this line in the reviewed manuscript by (lines 271-274):

"The expression of the inflammatory markers in mice after infection with the CV8 strain
was comparable that in the PBS-treated control mice (Fig. 6C)" -"...as a drug factory that is easy to remove with antibiotics once its action is completed." This statement should be revised. No data is presented supporting the efficacy of antibiotics in removing CV8 and related strains from the lungs. Antibiotic treatment should not be considered as a biocontainment strategy due to the undesired effects that it might have i.e., emergence of resistant variants, dysbiosis, etc. Further discussion of an effective biocontainment strategy for this lung-targeted living therapeutic should be included.
We understand the reviewer's concern. Recently, gene switches to limit M. pneumoniae growth (biosafety) for biomedical applications have been developed (https://doi.org/10.1038/s41467-022-29574-0). The biocontainment system described in that paper could be implemented in our chassis CV8 without potentially impacting the synthesis capacity of the product. We have included this in the discussion (lines 303-306).
In fact, gene switches to limit M. pneumoniae growth for biosafety applications have been developed (Broto et al. 2022). The biocontainment system described in that paper could be implemented in our chassis CV8 without potentially impacting the synthesis capacity of the product.
Reviewer #3: We would like to thank you for your critical revision of our manuscript ("Bacterial expression of a designed single-chain IL-10 prevents lung severe inflammation"). We pneumoniae live biotherapeutic chassis was presented. The key difference is the molecule (IL-10) that was secreted and the organ that is treated (lung) while the previous work was focussing on biofilm and catheters.
Overall, I found the work to be interesting and well performed. However, some sections can be confusing and would require rewording or clarifications. I would encourage the authors to use line numbers in future manuscripts to facilitate the review process.
1) Rewording a few sentences would enhance clarity and avoid misinterpretation.
-Activity of IL-10 derivatives; a few sentences suggest that the activity of the singlechain IL-10 shows a 60-fold increase in activity (abstract line 9; page 13, line 12. This can be misleading as the result is a combination of increased secretion by Mycoplasma pneumoniae and better activation of the receptor. There is no head-to-head comparison of purified single-chain IL-10 derivative with WT IL-10 at the same concentration that would support this conclusion. These sentence should be reworded to avoid any misunderstanding.
Indeed, it is true that for the HEK-Blue TM cells, the "60-fold" reflects a combination of expression and change in affinity. However, when looking at the FACS analysis, we see a 29-fold improvement in EC-50 for MutSC1, and a 57-fold improvement for MutSC2 in BLAeR cells. We have slightly modified the abstract to make this clearer.
'As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10-to 60-times, depending on the cell line' However, the reviewer is right that the sentence in Page 13, line12 is not correct Combining the higher expression and the better EC-50 shown in the HEK-Blue TM cell line, the WT Mycoplasma strain expressing MutSC2 has around 60 times relatively higher relative affinity in vitro than the IL-10 ORF strain Thus we have replaced it by (lines 333-336): 'Combining the higher expression and the better EC-50 shown in the HEK-Blue TM cell line, the supernatant of the WT Mycoplasma strain expressing MutSC2 has around 60times higher effect in vitro on HEK-Blue TM cells than the supernatant of the IL-10 ORF strain'.
-page 3, line 21. The sentence about the selection of a live biotherapeutic chassis with regards to the organ to treat could be reworded to improve syntax and clarity.
To improve the clarity of this point, we have re-structured the text. Now it appears as following in lines 41-46: 'The use of bacteria as delivery vectors or live biotherapeutic (LB) has the advantage of being able to produce the therapeutic agent locally, which lowers the required administered dose, thereby reducing the adverse effects and the production costs.
Recently, IL-10 expressed by Lactococcus lactis has been shown to be effective in preventing and treating colitis in a preclinical mice model (Steidler et al, 2000a;Cardoso et al, 2018) and also in phase I clinical trials of Crohn's disease (Braat et al, 2006).' Tables is difficult to understand without   additional  details: -page 12, second paragraph, lines 7-8: it is not obvious to me that the lung is more challenging than the intestine for live biotherapeutics. The authors could clarify their thoughts. I believe they meant that fewer bacteria are present in this organ but there are other factors to consider and it is not as simple as this.

2) the information in certain Supplementary
We agree with this comment and have now modified the corresponding discussion section (lines 317-320): We apologize for the confusion. Protein expression can vary across biological conditions because of differences in biomass or incubation time. Plus, the method used for biomass determination (CFU) or IL-10 quantification (ELISA) has a certain degree of variability between samples that increases when the production of IL-10 in fg per cell (CFU) is calculated. In the previous manuscript, we selected the number of molecules per cell for MutSC1 obtained in the replicate 'R2' as a correction factor to standardize the results. However, we believe that the normalization by the protein production per CFU (fg IL-10/CFU) of the reference IL-10 ORF is more appropriate. For this, we have now ,calculated the ratio ORF/ORF (=1), Mut3/ORF and MutSC1/ORF for each experiment (see new Table EV1). We observed that in all the experiments, the production of MutSC1 is 2.96x, 13.29x or, 2.25x of that produced from protein ORF, while the Mut3 production was comparable to the ORF in all the replicas analyzed. We have now modified panel B in the new Fig. 4 accordingly.
-Supplementary Table 2: why is the format of the data different in the "sulfide MutSC1" tab compared to "Sulfide WT IL-10" and "Sulfide MutSC2"?
The goal of the experiment is to identify the disulfide bridges in the WT IL-10, MutSC1 and MutSC2 proteins.
In the first experimental setting using MS, we clearly identified the cysteine12 that is carbamylated in IL-10 WT or MutSC2. However, this could not be precisely identified for MutSC1 (see Table EV2), likely due to the fact that peptides obtained after enzyme digestion were not optimal for MS detection. To improve this result, we performed an additional experiment for the MutSC1 to double-confirm the presence of carbamidomethylate in the first cysteine (Cys12) using PRM (Parallel Reaction Monitoring) chromatography followed by MS. This is the reason for the different formats of describing these (e.g., "Sulfide WT IL-10", "Sulfide MutSC2'', and "Sulfide MutSC1") in the previous version. We have now reorganized the data in the new Supplementary Table EV2 in the following sheets: "Sulfide WT IL-10 MS", "Sulfide MutSC2 MS", "Sulfide MutSC1 MS" and "Sulfide MutSC1 PRM + MS". Minor: -page 11, last line: 2 ug instead of 2 ugr.
-page 15, first sentence of the last paragraph: ATCC instead of ATTC.
We thank the reviewer for seeing these and have now corrected them.
-page 25, line 6. Bmax should be defined at first use.
We agree and have changed it.
-Supplementary Table 6: the "Inflammatory makers" tab should be "Inflammatory markers" We have corrected it.
-Supplementary Table 10: why is only the amino acid sequence provided for IL-10 derivatives? DNA sequences would ideally also be reported.
We agree and have now modified Supplementary Table EV9 to provide the DNA sequence plus the amino acid sequence and the specific details for each mutant.